Project Summary Pathogen enrichment by fragment size selection of cell-free DNA Cell-free DNA sequencing of plasma and sterile body fluids is an attractive approach for broad-based pathogen detection. Plasma cfDNA can sample a variety of body sites and allows for predictable and rapid (24-hour) results for agnostic pathogen detection, while existing screening regimens such as blood culture require 5 days or more to return negative. A major issue with shotgun sequencing approaches to infectious disease diagnostics is the high host background induced by inflammation can significantly compromise the analytical sensitivity of the assay. Human cfDNA forms a stereotyped fragment size distribution with a mode of 167 bp with fewer than ~5% of fragments measuring under 120 bp. Pathogen cfDNA is often overwhelmingly found in these shorter fragments as they rarely protect their DNA with nucleosomes. Here, we propose to optimize methods to selectively sequence short cfDNA fragments to increase the analytical sensitivity of cfDNA sequencing for pathogen detection. Specifically, we will optimize plasma extraction, library preparation, and size selection parameters to recover shorter fragments and deplete fragments > 120bp. We will then validate this approach on a series of 150 plasma specimens known to be positive for pathogens covering DNA viruses, bacteria, fungi/molds as well as 100 negative plasma specimens. Using this approach, our aim is to extend the analytical sensitivity of cfDNA sequencing to surpass that of specific qPCR approaches.